Molecular chaperones - Hsp90, Hsp70 and an assortment of cochaperones - play critical roles in steroid receptor function. Understanding the mechanisms by which chaperones establish, stabilize, and regulate the functional state of steroid receptors will provide important information on the basic biology of steroid receptors and of molecular chaperones. This proposal addresses critical gaps in our knowledge of how cochaperones cooperate with Hsp90 and Hsp70 to promote the progressive assembly and functional maturation of steroid receptor complexes. The experimental goal is to determine how the cochaperones Hip and Hop, which assemble transiently with Hsp-steroid receptor complexes at intermediate steps of the assembly pathway, recognize Hsp70 and Hsp90 and facilitate structural/functional transitions in receptor complexes. Structural and biochemical approaches in vitro will be combined with genetic approaches in a yeast model for steroid receptor function to gain physiological insight into the mechanisms and importance of cochaperone function. Specific Aim 1 - Establish how Hip enhances glucocorticoid receptor (GR) function in vivo. Preliminary results show that Hip significantly enhances hormone-dependent expression of a GR reporter gene. In this aim, we will construct a stable GR reporter strain, determine how GR is altered by Hip, and test what domains of Hip are required to increase GR signaling. Specific Aim 2 - Establish how Hop binds Hsp70 and Hsp90 and how these interactions relate to chaperone-mediated enhancement of GR function in vivo. Current models for Hop interactions with Hsp70 and Hsp90 cannot explain several experimental observations. We will test the hypothesis that domain interactions of Hop are critically important for Hsp binding and Hop function in cells. Conclusions from Hop mutational studies will be confirmed by X-ray crystallography of Hop-Hsp complexes. Specific Aim 3 - Clarify in vivo the roles of Hsp90, Hsp70, and cochaperone interactions in GR assembly. The highly conserved EEVD sequences that terminate both Hsp90 and Hsp70 have been strongly implicated in cochaperone binding, yet there are genetic and biochemical observations that conflict with this view. We aim to resolve the cellular importance of EEVD and other cochaperone interaction sites on Hsp90 and Hsp70 by gene replacements in yeast. [unreadable] [unreadable]